山慈菇水提物抗肝癌有效部位筛选及其对肝癌小鼠肿瘤血管生成的影响  

Screening of anti-hepatocarcinoma active fraction from water extract of Cremastrae Pseudobulbus Pleiones Pseudobulbus and its effect on tumor angiogenesis in mice with hepatocarcinoma

作　　者：芦艺凡 王宇 姜婷月 李佳园 范文昕 张鸽 石晋丽[1] LU Yifan;WANG Yu;JIANG Tingyue;LI Jiayuan;FAN Wenxin;ZHANG Ge;SHI Jinli(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药学院,北京102488

出　　处：《中草药》2025年第5期1607-1616,共10页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(82073971)。

摘　　要：目的明确山慈菇Cremastrae Pseudobulbus Pleiones Pseudobulbus水提物的抗肝癌主要有效部位,探讨其有效部位对肝癌小鼠肿瘤血管生成的作用及机制。方法采用正交试验优化山慈菇水提物提取条件,醇沉法将水提物分为醇沉部位与醇溶部位,苯酚硫酸法检测各部位多糖含量,在药效指导下进行有效部位筛选。将60只balb/c小鼠随机分为空白组、模型组、索拉非尼组(30 mg/kg)、山慈菇水提物组、山慈菇醇沉物组及山慈菇醇溶物组(均以生药量计1.82 g/kg),每组10只,连续ig给药16 d。通过计算抑瘤率及苏木精-伊红(hematoxylin eosin,HE)染色观察各组肿瘤组织病理变化综合评价药效;取有效部位组小鼠肿瘤组织或血清,原位末端标记(terminal deoxynucleotidyl transferase dUTP nick-end labeling,TUNEL)法测定肿瘤细胞凋亡情况;酶联免疫吸附试验(enzyme-linked immunosorben assay,ELISA)检测小鼠血清中血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-2(matrix metallopeptidase-2,MMP-2)和MMP-9水平;免疫组织化学法(immunohistochemistry,IHC)染色观察肿瘤组织微血管生成情况;Western blotting法检测肿瘤组织VEGF、MMP-2、MMP-9以及Wnt1、β-连环蛋白(β-catenin)的表达变化。结果正交试验优化得山慈菇水提物最优提取条件为浸泡时间90 min、料液比1∶30、提取时间120 min、提取次数2次,山慈菇水提物、醇沉物、醇溶物得率分别为37.76%、28.00%、5.53%,含量测定得多糖含量分别为70.18%、95.19%、0.58%。动物实验结果显示,山慈菇水提物组、山慈菇醇沉物组、山慈菇醇溶物组抑瘤率分别为43.91%、43.39%、21.60%;病理切片显示,模型组肿瘤组织病变显著,各给药组均有所改善,以山慈菇水提物组与醇沉物组胞核碎裂更为明显。与模型组比较,山慈菇醇沉物组肿瘤细胞凋亡率显著增加(P<0.01);小鼠血清与肿瘤组织中VEGF、MMP-2、MMP-9水平显著降低(P<0.Objective To identify the primary anti-hepatocarcinoma active site in Shancigu(Cremastrae Pseudobulbus Pleiones Pseudobulbus)aqueous extract and investigate its effects and mechanisms on tumor angiogenesis in mice with liver cancer.Methods After optimizing the extraction conditions of Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract through orthogonal experiment,the aqueous extract was divided into alcohol precipitation and alcohol soluble parts using alcohol precipitation method,phenol-sulfuric acid method was used to detect the content of polysaccharides in each part,and effective parts were screened under the guidance of pharmacological effects.A total of 60 BALB/c mice were randomly assigned into a blank group,a model group,a sorafenib group(30 mg/kg),a Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract group,a Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments group,and a Cremastrae Pseudobulbus Pleiones Pseudobulbus alcohol soluble substance group(all calculated based on the amount of raw medicine 1.82 g/kg),with 10 mice in each group.The mice were intragastrically administered continuously for 16 d.The drug efficacy was comprehensively evaluated by calculating tumor inhibition rate and observing pathological changes in tumor tissues of each group through hematoxylin-eosin(HE)staining.Tumor tissue or mouse serum after effective site administration were selected,then terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay was performed to detect apoptosis in tumor cells.Enzyme-linked immunosorben assay(ELISA)was used to determine the levels of vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2),and MMP-9 in mice serum.Immunohistochemical(IHC)was used to observe micro vessel density(MVD)in tumor tissues.Western blotting was used to detect the expression changes of VEGF,MMP-2,MMP-9,Wnt1 andβ-catenin.Results The optimal extraction conditions were 90 min of soaking time,a material-to-liquid ratio of 1:30,120 min of extraction time,and

关 键 词：山慈菇 山慈菇多糖 肝细胞癌 血管生成 WNT/Β-CATENIN通路 

分 类 号：R285.5[医药卫生—中药学]